Reactive oxygen species (ROS) can be induced by both endogenous and exogenous sources and they can result in DNA damage. We hypothesize that the ROS-induced bulky DNA lesions, owing to their difficulty in being repaired and their miscoding potential, may contribute significantly to the ubiquitously observed mutations at CpG dinucleotide site. To test this hypothesis, we will take an interdisciplinary approach, which encompasses synthetic organic chemistry, bioanalytical chemistry and molecular biology, to investigate the formation of these lesions in vitro and in vivo and to assess how they are interpreted by DNA replication and repair machineries in human cells. We organize the proposed research into three specific aims: In Aim 1, we will assess quantitatively the formation of bulky lesions in DNA upon exposure to Fenton reagents and investigate how cytosine methylation affects the formation of 8,5'-cyclo-2'-deoxyguanosine at CpG site. We will also quantify these lesions formed in tissues of a rat model of Wilson's disease, which arises from excessive accumulation of copper ion in the liver. In Aim 2, we will investigate the cytotoxic and mutagenic properties of these lesions in mammalian cells by using our newly developed shuttle vector technology. In Aim 3, we will examine how the ROS-induced bulky DNA lesions are repaired in human cells. The significance of the proposed research lies in that it will provide important insights into the implications of these lesions in the development of human diseases including cancer and neurological disorders. Additionally, the results from the proposed research may reveal potential risk factors for developing human diseases and lead to the discovery of new molecular signatures for monitoring the human exposure toward ROS. The proposed research is highly innovative because it involves the use of a multi-pronged approach to tackle an important, yet largely overlooked biological problem.